1. Field of the Invention
The present invention relates to novel metal-ligand amino acid derivatives which are useful as Magnetic Resonance Imaging (MRI) agents or as compounds useful to incorporate a metal ion containing-chelate amino acid derivative at any point in a polypeptide during conventional solid phase synthesis of a polypeptide.
2. Description of the Related Art
MRI contrast enhancing agents
The utility of magnetic resonance imaging (MRI) also known as nuclear magnetic resonance (NMR) imaging in diagnostic medicine has recently been improved by the development of pharmaceutical MRI contrast agents which change the relaxation times of water protons in the vicinity of the agent. A pharmaceutical MRI contrast agent is selected to bind to a component of a body tissue under study, thereby increasing the relaxivity of water protons in the vicinity of the tissue to which the agent is bound. Thus, the MRI signal from the tissues of interest is enhanced relative to the surrounding tissues. MRI contrast image enhancing agents incorporate organic groups into metal chelating ligands to produce metal ion-chelate MRI contrast enhancing agents which preferentially bind to specific proteins in a non-covalent and non-immunologic manner. As a result of this binding the protons of the water molecules in the vicinity of the metal ion chelates have a relaxivity that is enhanced by at least a factor of two or more relative to the relaxivity induced by the paramagnetic complex free in solution.
Tissue specificity of MRI contrast agents is due in part to the structure of the metal ion chelate and its ability to mimic the structure of naturally occurring molecules which have an affinity for the tissue of interest (e.g. liver, gall bladder intestine, heart). Further, the binding of the metal ion chelates to such tissues is sometimes enhanced by the incorporation of substituents which increase the lipophilicity and hydrophobicity of specific portions of the molecule.
Some of the metal ion chelates mimic the structure of bilirubin and thereby exhibit preferential binding to albumin, to the hepatocellular uptake protein, to ligandin, and to the fatty acid binding proteins. The ability of the organic chelates to bind to these proteins renders them useful in enhancing the image of normal liver tissue on the presence of tumors, for monitoring liver function, and for enhancing the image of the bile ducts and gall bladder. In addition, binding to albumin in the blood creates a high relaxivity blood-pool contrast agent that is useful in detecting disruption of the blood-brain barrier, in MRI angiography, in perfusion imaging, and in distinguishing between tumors and blood-filled lesions such as hemangiomas and hemorrhage.
Some references of interest in the synthesis and of these novel amino acid derivatives useful as MRI contrast enhancing agents are as follows:
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Peptide Protein Res. 1990, Vol. 35, 161-214. PA1 11.(a) J. C. Sheehan, et al. J. Org. Chem 1964, Vol. 29, 2006-2008. 11(b). G. C. Stelakatos, et al. J. Chem. Soc. 1966, C, pp. 1191-1199. PA1 12. S. S. Isied, et al. J. Am. Chem. Soc. 1982, Vol. 104. 3910-3916. PA1 13. E. Atherton, et al. J. Chem. Soc. Chem. Com. 1978, p. 537, and 539. PA1 14. L. A. Carpino, et al. J. Org. Chem. 1972, Vol. 37, 3404. PA1 (i) the metal binding moiety is not exactly that of EDTA and affinity cleavage of proteins using this chelate (ethylenediaminetriacetic acid) results in non-hydrolytic fragments, (Ref. 3), and PA1 (ii) stability of metal complexes may be a problem during the use of the chelate to prepare radiopharmaceuticals. PA1 (a) repetitive TFA acidolysis in Boc-group deprotection could lead to acid catalyzed side-reactions, and PA1 (b) cleavage and deprotection of peptides requires HF and specific laboratory set up which is not available to many researchers. Due to these concerns Fmoc (9-fluorenylmethylcarbamate) solid phase peptide synthesis was developed which employs N-.alpha.-Fmoc amino acid (Ref. 10). In this polypeptide approach, the Fmoc group is deprotected with piperidine and trifluoroacetic acid (TFA) is required only for the final cleavage and deprotection step. Compound 4 of this invention was designed to be compatible with the Fmoc solid phase peptide synthesis strategy. PA1 Q is selected from a straight chain alkylene, branched chain alkylene, or alicyclic alkylene having 1 to 10 carbon atoms, phenylene, ##STR2## Q .sup.1 is independently selected from --OH, OCH.sub.3, OCH.sub.2 CH.sub.3, O-phenyl, O-benzyl, NH.sub.2, NHCH.sub.3, NHCH.sub.2 CH.sub.3 N(CH.sub.3).sub.2, or N(CH.sub.2 CH.sub.3).sub.2. PA1 Z.sup.1, Z.sup.2, Z.sup.3 and Z.sup.4 are each independently selected from --CH.sub.2 --, --CH.sub.2 CH.sub.2 --, --CH.sub.2 --CH(CH.sub.3)--CH.sub.2 --, or --CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 --; PA1 R.sup.1, R.sup.2 R.sup.3, and R.sup.4 are each independently selected from --H, --C(CH.sub.3).sub.3 or when R.sup.1, R.sup.2, R.sup.3, and R.sub.4 are in ion form they coordinate as bonds to a metal ion PA1 when Y is ##STR3## A is an amine protecting group selected from Fmoc or other protecting groups provided that it is not t-Boc; PA1 Z.sup.5 is independently selected from Z.sup.1 and PA1 R.sup.5 is independently selected from R.sup.1 or when in ion form R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 coordinate a metal ion M as shown PA1 (A) contacting a compound of the structure: ##STR4## wherein Y is in a direct bond or --CH.sub.2 NHCH.sub.2 -- with sufficient compound of the structure X--Z.sup.1 --(C.dbd.O)OC(CH.sub.3).sub.3, PA1 wherein X is halogen selected from chloro, bromo or iodo, and PA1 Z.sup.1 is selected from --CH.sub.2 -- or --CH.sub.2 CH.sub.2 -- to react with all --NH bonds present in sufficient dipolar aprotic solvent to cause dissolution of II and in the presence of a tertiary alkyl amine; PA1 (B) refluxing the reaction mixture of step (A) for between about 5 and 48 hr followed by cooling to ambient temperature and removing the solvent under vacuum producing crude compound III; ##STR5## and Z.sup.5 is defined above; (C) contacting the product remaining in step (B) with an anhydrous water-immiscible oxygenated alkyl containing solvent with stirring to redissolve the product; PA1 (D) separating and drying the water immiscible organic phase, adding a polar aprotic water immiscible solvent; PA1 (E) washing the organic phase with an aqueous buffer having a pH of between about 1 and 4; PA1 (F) separating the organic phase and aqueous phase; PA1 (G) drying the organic phase and removing the solvent in vacuum producing a purified polyaminepolyester having multiple --C(CH.sub.3).sub.3 ester groups of structure III; PA1 (H) dissolving the product of step (G) in sufficient dimethylformamide, diethylformamide, hexamethylphosphoramide, tetramethylenesulfone, dimethylsulfoxide or mixtures thereof adding sodium thiophenoxide to produce Compound III wherein Q.sup.1 is OH; PA1 (I) heating to between about 90.degree. and 110.degree. C. for between about 0.5 and 6 hr, cooling to ambient temperature with dilution using a volume equivalent of a polar aprotic water-immiscible hydrocarbon solvent; PA1 (J) contacting the organic solution of step (I) with aqueous buffer having a pH between about 1 and 4, separating the organic layer and removing the solvent under vacuum; PA1 (K) purifying the product of step (J) by column chromatography using an eluent of increasing polarity of a mixture of n-hexane and ethyl acetate followed by a weak organic acid in an organic alkyl ester; PA1 (L) contacting the product of step (K), the polybutyl ester protected polyfunctional chelating agent; with a chlorinated hydrocarbon solvent and a carbonyl group activator selected from 1-hydroxybenzotriazole, HBTU, TBTU, or BOP, contacting at 0.degree. C. the solution of step (K) with a peptide coupling agent selected from carbodiimide, DCC, or CDI or EDC, followed by filtration, and evaporating the filtrate in vacuum; PA1 (M) contacting with stirring the activated ester of step (L) with excess compound of structure IV: EQU N-.alpha.-Fmoc-NH--CH--(COOH)--Q--NH.sub.2 IV PA1 at ambient temperature for between 6 and 48 hr in oxygenated organic water immiscible liquid and a tertiary alkyl amine to adjust the pH of the reaction to between about 7 and 9, followed by removal of the solvent in vacuum; PA1 (N) dissolving the product of step (M) in an organic ester to remove excess protected amino acid in the presence of a weak organic polyacid, drying the solution and removal of the solvent under reduced pressure; PA1 (O) purifying the crude product of step (N) using reverse phase; chromatography producing the amino acid-chelate of structure I. PA1 (h) contacting the polypeptide chelate produced herein with aqueous buffer at pH 6 to 8 at ambient temperature adding aqueous metal M salts to produce the metal ion chelate and isolating the metal ion chelate.